Many lipids or lipid-derived products generated by phospholipases acting on phospholipids in membranes have been implicated as mediators and second messengers in signal transduction. Lipid-derived second messengers of signal transduction are typically short-lived lipid metabolites that are synthesized from membrane-derived lipid precursors in response to cell stimulation (e.g., ligand-receptor coupling, electrical stimulation, and elevation in intracellular calcium). The production of lipid-derived second messengers is initiated by the activation of intracellular phospholipases, which liberate metabolites capable of propagating a cell-specific cascade of biochemical events that collectively results in cell activation.
One such lipid second messenger is arachidonic acid which is found esterified in the sn-2 position of membrane phospholipids and can potentially be released by a number of phospholipases, although recent research points to phospholipase A.sub.2 as the major mediator of arachidonic acid release, at least for prostaglandin and leukotriene biosynthesis. The other product of phospholipase A.sub.2 action is lysophospholipid, a class of amphiphilic molecules that contribute to membrane regulation by virtue of their ability to alter membrane physical properties and also serve either as direct precursors of lipid second messengers (e.g., platelet activating factor) or are second messengers in their unmodified form. When the phospholipid is an alkyl ethercontaining phosphatidylcholine, the lysophospholipid upon acetylation forms platelet-activating factor (PAF), another lipid that is a potent cellular mediator.
Arachidonic acid mobilized from intracellular phospholipid storage depots in cellular membranes thus has multiple metabolic fates including: 1) internal oxidation, resulting in the generation of biologically active eicosanoids; 2) transacylation, resulting in the redistribution of arachidonic acid in phospholipid molecular species; 3) thioesterification, resulting in the subsequent reincorporation of released arachidonic acid into lipid metabolic pathways; or 4) secretion into the extracellular space, facilitating cell to cell communication.
Davidson and Dennis (1990) J. Mol. Evol. 31, 228-238 recently compared and aligned all of the known sequences of phospholipase A.sub.2. Low molecular weight phospholipases A.sub.2 sequenced to date are all considered to be secreted and are composed of a single polypeptide chain, about 120 amino acids long, containing 10-14 cysteines, all in disulfide pairs. These cysteines constitute the bulk of the sequence conservation between the mammalian, reptile and insect secreted enzymes. In addition, these phospholipases A.sub.2 require Ca.sup.+2 for activity and contain a conserved Ca.sup.+2 binding loop, whereas the nearby catalytic site contains a histidine/aspartic acid pair conserved throughout. Two human low molecular weight phospholipases A.sub.2 have been sequenced. One is from the pancreas and is similar to the venom phospholipases A.sub.2 of the old-world cobras and kraits, except for the addition of an internal loop of five amino acids and the fact that it is produced as a proenzyme. The other sequenced human low molecular weight phospholipase A.sub.2, originally isolated from platelets and synovial fluid, is similar to the venom phospholipases A.sub.2 of old- and new-world snakes such as the diamondback rattlesnake. These sequences have an extended COOH terminus and a related but distinct disulfide bond pattern. Bee venom phospholipase A.sub.2 shows a highly divergent sequence that is missing an NH.sub.2 -terminal section, but is homologous in other regions.
In order to fully appreciate the activity of phospholipase A.sub.2 and its role in cellular communication and disease, much research has been done on extracellular secreted phospholipases A.sub.2. Although these studies have provided valuable information, the contribution of intracellular phospholipase A.sub.2 has not been well-studied. The single human non-low molecular weight phospholipase A.sub.2 sequenced to date is an 85kD polypeptide which shares limited structural homology with protein kinase C, GAP, and phospholipase C, Clark et al. (1991) Cell 65, 1043-1051 and Sharp et al. (1991) J. Biol. chem. 266, 14850-14853. Other forms of intracellular phospholipase A.sub.2 remain poorly characterized. Thus there continues to be a need for identification and characterization of new phospholipase A.sub.2 enzymes.